5 strand resection at DNA double strand breaks (DSBs) is critical

Published January 22, 2018

5 strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. lead to chromosome rearrangements, genomic instability and tumorigenesis if not repaired correctly (1). Nonhomologous end joining (NHEJ) and homologous recombination (HR) are the two major DSB repair pathways in eukaryotic cells. NHEJ is the primary pathway and is used throughout the cell cycle, while HR is active in S and G2 phases where sister chromatids are available as repair templates (2). HR is initiated with the resection of the 5 strands to generate 3 single-stranded DNA (ssDNA), buy TGX-221 which is required for Rad51 binding and strand invasion. Therefore, the initiation of resection is thought to be a critical control point for the choice between HR and NHEJ because this process commits the breaks to HR repair. The proteins required for 5 strand resection at DSBs in eukaryotic cells include the Mre11/Rad50/Nbs1 (MRN) complex, which binds to DSBs and promotes resection by two independent endo/exonucleases: Exo1 and Dna2 (3). The CtIP protein also participates in this process and promotes long-range resection in conjunction with MRN (4). The Ataxia-Telangiectasia-Mutated (ATM) protein kinase has also been shown to be required for DSB resection (5C7) although its role in this process is not completely understood. The Ku70/80 heterodimer and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) coordinate the process of NHEJ. After binding of Ku and subsequent recruitment of DNA-PKcs to DSBs, an active DNA-PK holoenzyme is buy TGX-221 formed that mediates the phosphorylation of DNA-PKcs itself as well as other NHEJ buy TGX-221 factors (8). Autophosphorylated DNA-PKcs undergoes a large conformational change that is thought to promote its dissociation from DNA ends and facilitate end joining (9C13). Residues T2609 and T2647 in the SORBS2 ABCDE cluster are DNA-PKcs autophosphorylation sites (14) but have also been shown to be targets of ATM (15). Apart from its role in NHEJ, DNA-PKcs has also been implicated in regulation of HR (13,16C19), but the underlying mechanism is not fully understood. Previous studies have demonstrated that the human Ku heterodimer inhibits 5 strand resection mediated by Exo1 or Dna2 system To quantitate ssDNA at sites of DSBs, we used the ER-(20). Consistent with these observations, we found that depletion of SOSS1 complex using siRNA directed against the SOSS-A subunit led to reduced resection in U2OS cells, as did the depletion of Exo1 (Figure 3C and D), one of the two major exonucleases involved in long-range resection of DSB ends. There is some variability in the buy TGX-221 ssDNA% at DSB1 and DSB2 between different experiments, which can be partially attributed to the variability in break formation by AsiSI (Supplementary Table S1). Figure 3. The effects of known DNA repair factors on resection. Measurement of DSB resection in ER-and find that phosphorylation of DNA-PKcs is essential for resection when DNA-PK is present at DNA ends (61). We conclude that the DNA-PKcs protein inhibits resection, but this inhibition can be overcome by DNA-PKcs phosphorylation that promotes dissociation of DNA-PKcs kinase from DSB ends and recruitment of resection enzymes. Using the AsiSI system shown here we also measured the levels of DSBs and observed increased accumulation of unresolved breaks on 4-OHT treatment in Ku or DNA-PKcs deficient cells (Supplementary Table S1), likely due to a failure of NHEJ repair. Notably, even though depletion of 53BP1 and depletion of Ku or DNA-PKcs have similar stimulatory effects on resection, only depletion of the DNA-PK holoenzyme components increases the apparent accumulation of unresolved DSBs (Supplementary Table S1). This suggests that 53BP1 is not involved in the initial fast process of religation but may affect pathway choice for the subset of breaks that goes through resection and HR. Recent observations showing that 53BP1 deletion rescues many of the defects seen in BRCA1-deficient cells (42,43) suggest that BRCA1 function must be to antagonize 53BP1 in some way. Yet here we demonstrate that BRCA1 depletion has minimal effect on end processing under conditions where Rad51 filament formation is clearly compromised. It is possible that Rad51 buy TGX-221 filaments in BRCA1-depleted cells are qualitatively different from those in WT cells, leading to.